Many symbiotic or pathogenic bacteria have evolved complex specialized machines known as type III secretion systems (T3SS), which have the capacity to transfer multiple bacterially-encoded proteins into host eukaryotic cells. These machines are of great interest because they are central to the pathogenic or symbiotic relationships of the bacteria that encode them. Proteins delivered by these machines, collectively referred to as effectors, can modulate a great variety of eukaryotic cellular functions to shape the bacteria/host cell functional interphase. Through work supported by this Grant, we have been studying a T3SS from Salmonella enterica serovar Typhimurium (S. Typhimurium), encoded within its pathogenicity island 1 (SPI-1). This system mediates several phenotypes that are essential for virulence including bacterial entry into and survival within non-phagocytic cells, the induction of programmed cell death in macrophages, and the modulation of innate immune responses and inflammation in the intestinal tract. The central component of all T3SSs is the needle complex, a supramolecular structure which allows the translocation of proteins through the bacterial envelope during their journey to eukaryotic target cells. The needle complex works in association with other components such as the export apparatus, which mediates passage of the secreted proteins through the bacterial inner membrane, and the sorting platform, a cytoplasmic structure that establishes an order in the secretion process. Additional components regulate the function of this machine to ensure that it is activated and deployed at the appropriate time (i.e. upon contact with target cells). Energy to drive protein translocation and the unfolding of the protein substrates is derived from an associated ATPase as well as a proton gradient established by poorly understood mechanisms. This research project intends to investigate less understood aspects of type III protein secretion including the mechanism of signal transduction through the needle substructure, the topological organization of the sorting platform and export apparatus, and the function of the type III secretion associated ATPase. Accomplishing these objectives will not only enhance our understanding of Salmonella spp. pathogenesis but also our understanding of T3SSs in general. Since this system is central to the pathogenesis of many important pathogenic bacteria, these studies may provide the bases for the development of broadly applicable anti-infective strategies.